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1.
Int J Mol Sci ; 22(4)2021 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-33672263

RESUMO

The 70 kDa and 90 kDa heat shock proteins Hsp70 and Hsp90 are two abundant and highly conserved ATP-dependent molecular chaperones that participate in the maintenance of cellular homeostasis. In Escherichia coli, Hsp90 (Hsp90Ec) and Hsp70 (DnaK) directly interact and collaborate in protein remodeling. Previous work has produced a model of the direct interaction of both chaperones. The locations of the residues involved have been confirmed and the model has been validated. In this study, we investigate the allosteric communication between Hsp90Ec and DnaK and how the chaperones couple their conformational cycles. Using elastic network models (ENM), normal mode analysis (NMA), and a structural perturbation method (SPM) of asymmetric and symmetric DnaK-Hsp90Ec, we extract biologically relevant vibrations and identify residues involved in allosteric signaling. When one DnaK is bound, the dominant normal modes favor biological motions that orient a substrate protein bound to DnaK within the substrate/client binding site of Hsp90Ec and release the substrate from the DnaK substrate binding domain. The presence of one DnaK molecule stabilizes the entire Hsp90Ec protomer to which it is bound. Conversely, the symmetric model of DnaK binding results in steric clashes of DnaK molecules and suggests that the Hsp90Ec and DnaK chaperone cycles operate independently. Together, this data supports an asymmetric binding of DnaK to Hsp90Ec.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Regulação Alostérica , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
2.
Nat Commun ; 12(1): 828, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547294

RESUMO

The co-chaperone p23 is a central part of the Hsp90 machinery. It stabilizes the closed conformation of Hsp90, inhibits its ATPase and is important for client maturation. Yet, how this is achieved has remained enigmatic. Here, we show that a tryptophan residue in the proximal region of the tail decelerates the ATPase by allosterically switching the conformation of the catalytic loop in Hsp90. We further show by NMR spectroscopy that the tail interacts with the Hsp90 client binding site via a conserved helix. This helical motif in the p23 tail also binds to the client protein glucocorticoid receptor (GR) in the free and Hsp90-bound form. In vivo experiments confirm the physiological importance of ATPase modulation and the role of the evolutionary conserved helical motif for GR activation in the cellular context.


Assuntos
Adenilil Imidodifosfato/química , Proteínas de Choque Térmico HSP90/química , Chaperonas Moleculares/química , Prostaglandina-E Sintases/química , Receptores de Glucocorticoides/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Adenilil Imidodifosfato/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Simulação de Dinâmica Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Prostaglandina-E Sintases/genética , Prostaglandina-E Sintases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
3.
Nat Commun ; 12(1): 946, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33574241

RESUMO

The Hsp40/Hsp70 chaperone families combine versatile folding capacity with high substrate specificity, which is mainly facilitated by Hsp40s. The structure and function of many Hsp40s remain poorly understood, particularly oligomeric Hsp40s that suppress protein aggregation. Here, we used a combination of biochemical and structural approaches to shed light on the domain interactions of the Hsp40 DnaJB8, and how they may influence recruitment of partner Hsp70s. We identify an interaction between the J-Domain (JD) and C-terminal domain (CTD) of DnaJB8 that sequesters the JD surface, preventing Hsp70 interaction. We propose a model for DnaJB8-Hsp70 recruitment, whereby the JD-CTD interaction of DnaJB8 acts as a reversible switch that can control the binding of Hsp70. These findings suggest that the evolutionarily conserved CTD of DnaJB8 is a regulatory element of chaperone activity in the proteostasis network.


Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Evolução Biológica , Células HEK293 , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP70/química , Humanos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Proteostase , Especificidade por Substrato
4.
Int J Mol Sci ; 22(4)2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33557244

RESUMO

Cell surface and secreted proteins provide essential functions for multicellular life. They enter the endoplasmic reticulum (ER) lumen co-translationally, where they mature and fold into their complex three-dimensional structures. The ER is populated with a host of molecular chaperones, associated co-factors, and enzymes that assist and stabilize folded states. Together, they ensure that nascent proteins mature properly or, if this process fails, target them for degradation. BiP, the ER HSP70 chaperone, interacts with unfolded client proteins in a nucleotide-dependent manner, which is tightly regulated by eight DnaJ-type proteins and two nucleotide exchange factors (NEFs), SIL1 and GRP170. Loss of SIL1's function is the leading cause of Marinesco-Sjögren syndrome (MSS), an autosomal recessive, multisystem disorder. The development of animal models has provided insights into SIL1's functions and MSS-associated pathologies. This review provides an in-depth update on the current understanding of the molecular mechanisms underlying SIL1's NEF activity and its role in maintaining ER homeostasis and normal physiology. A precise understanding of the underlying molecular mechanisms associated with the loss of SIL1 may allow for the development of new pharmacological approaches to treat MSS.


Assuntos
Suscetibilidade a Doenças , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Nível de Saúde , Chaperonas Moleculares/metabolismo , Animais , Biomarcadores , Gerenciamento Clínico , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Estudos de Associação Genética , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Humanos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutação , Fenótipo , Ligação Proteica , Conformação Proteica , Transdução de Sinais , Degenerações Espinocerebelares/diagnóstico , Degenerações Espinocerebelares/etiologia , Degenerações Espinocerebelares/metabolismo , Degenerações Espinocerebelares/terapia , Relação Estrutura-Atividade , Resposta a Proteínas não Dobradas
5.
J Mol Biol ; 433(5): 166826, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33453188

RESUMO

The folding of disulfide bond containing proteins in the endoplasmic reticulum (ER) is a complex process that requires protein folding factors, some of which are protein-specific. The ER resident saposin-like protein pERp1 (MZB1, CNPY5) is crucial for the correct folding of IgA, IgM and integrins. pERp1 also plays a role in ER calcium homeostasis and plasma cell mobility. As an important factor for proper IgM maturation and hence immune function, pERp1 is upregulated in many auto-immune diseases. This makes it a potential therapeutic target. pERp1 belongs to the CNPY family of ER resident saposin-like proteins. To date, five of these proteins have been identified. All are implicated in protein folding and all contain a saposin-like domain. All previously structurally characterized saposins are involved in lipid binding. However, there are no reports of CNPY family members interacting with lipids, suggesting a novel function for the saposin fold. However, the molecular mechanisms of their function remain elusive. To date, no structure of any CNPY protein has been reported. Here, we present the high-resolution (1.4 Å) crystal structure of human pERp1 and confirm that it has a saposin-fold with unique structural elements not present in other saposin-fold structures. The implications for the role of CNPY proteins in protein folding in the ER are discussed.


Assuntos
Imunoglobulina A/química , Imunoglobulina M/química , Chaperonas Moleculares/química , Saposinas/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Imunidade Humoral , Imunoglobulina A/genética , Imunoglobulina A/imunologia , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/imunologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Saposinas/genética , Saposinas/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
6.
Gene ; 774: 145420, 2021 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-33434627

RESUMO

ClpXP in Escherichia coli is a proteasome degrading protein substrates. It consists of one hexamer of ATPase (ClpX) and two heptamers of peptidase (ClpP). The ClpX binds ATP and translocates the substrate protein into the ClpP chamber by binding and hydrolysis of ATP. At single molecular level, ClpX harnesses cycles of power stroke (dwell and burst) to unfold the substrates, then releases the ADP and Pi. Based on the construction and function of ClpXP, especially the recent progress on how ClpX unfold protein substrates, in this mini-review, a currently proposed single ClpX molecular model system detected by optical tweezers, and its prospective for the elucidation of the mechanism of force generation of ClpX in its power stroke and the subunit interaction with each other, were discussed in detail.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/fisiologia , Endopeptidase Clp/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/enzimologia , Chaperonas Moleculares/fisiologia , Imagem Individual de Molécula , ATPases Associadas a Diversas Atividades Celulares/química , Pesquisa Biomédica , Endopeptidase Clp/química , Proteínas de Escherichia coli/química , Redes e Vias Metabólicas , Mitocôndrias/fisiologia , Modelos Moleculares , Chaperonas Moleculares/química , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/fisiologia , Estrutura Molecular , Relação Estrutura-Atividade
7.
Int J Mol Sci ; 22(3)2021 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-33513816

RESUMO

Mitochondrial homeostasis refers to the balance of mitochondrial number and quality in a cell. It is maintained by mitochondrial biogenesis, mitochondrial fusion/fission, and the clearance of unwanted/damaged mitochondria. Mitophagy represents a selective form of autophagy by sequestration of the potentially harmful mitochondrial materials into a double-membrane autophagosome, thus preventing the release of death inducers, which can trigger programmed cell death (PCD). Recent advances have also unveiled a close interconnection between mitophagy and mitochondrial dynamics, as well as PCD in both mammalian and plant cells. In this review, we will summarize and discuss recent findings on the interplay between mitophagy and mitochondrial dynamics, with a focus on the molecular evidence for mitophagy crosstalk with mitochondrial dynamics and PCD.


Assuntos
Apoptose/genética , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Mitofagia , Animais , Autofagossomos/genética , Autofagossomos/metabolismo , Homeostase , Mamíferos , Mitocôndrias/genética , Mitofagia/genética , Mitofagia/fisiologia , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Plantas , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Ubiquitina/genética , Ubiquitina/metabolismo , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
8.
Exp Cell Res ; 399(2): 112491, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33460589

RESUMO

HSP70 chaperones, J-domain proteins (JDPs) and nucleotide exchange factors (NEF) form functional networks that have the ability to prevent and reverse the aggregation of proteins associated with neurodegenerative diseases. JDPs can interact with specific substrate proteins, hold them in a refolding-competent conformation and target them to specific HSP70 chaperones for remodeling. Thereby, JDPs select specific substrates and constitute an attractive target for pharmacological intervention of neurodegenerative diseases. This, under the condition that the exact mechanism of JDPs interaction with specific substrates is unveiled. In this review, we provide an overview of the structural and functional variety of JDPs that interact with neurodegenerative disease-associated proteins and we highlight those studies that identified specific residues, domains or regions of JDPs that are crucial for substrate binding.


Assuntos
Proteínas de Transporte/metabolismo , Doenças Neurodegenerativas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Animais , Proteínas de Transporte/química , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Doenças Neurodegenerativas/patologia , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas/fisiologia , Mapas de Interação de Proteínas/fisiologia
9.
Methods Mol Biol ; 2209: 1-16, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33201459

RESUMO

Cellular RNAs depend on proteins for efficient folding to specific functional structures and for transitions between functional structures. This dependence arises from intrinsic properties of RNA structure. Specifically, RNAs possess stable local structure, largely in the form of helices, and there are abundant opportunities for RNAs to form alternative helices and tertiary contacts and therefore to populate alternative structures. Proteins with RNA chaperone activity, either ATP-dependent or ATP-independent, can promote structural transitions by interacting with single-stranded RNA (ssRNA) to compete away partner interactions and then release ssRNA so that it can form new interactions. In this chapter we review the basic properties of RNA and the proteins that function as chaperones and remodelers. We then use these properties as a foundation to explore key points for the design and interpretation of experiments that probe RNA rearrangements and their acceleration by proteins.


Assuntos
Proteínas , RNA , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Conformação de Ácido Nucleico , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas/química , Proteínas/metabolismo , RNA/química , RNA/metabolismo
10.
Biochim Biophys Acta Mol Cell Res ; 1868(1): 118881, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33022276

RESUMO

Heme, as a hydrophobic iron-containing organic ring, is lipid soluble and can interact with biological membranes. The very same properties of heme that nature exploits to support life also renders heme potentially cytotoxic. In order to utilize heme, while also mitigating its toxicity, cells are challenged to tightly control the concentration and bioavailability of heme. On the bright side, it is reasonable to envision that, analogous to other transition metals, a combination of membrane-bound transporters, soluble carriers, and chaperones coordinate heme trafficking to subcellular compartments. However, given the dual properties exhibited by heme as a transition metal and lipid, it is compelling to consider the dark side: the potential role of non-proteinaceous biomolecules including lipids and nucleic acids that bind, sequester, and control heme trafficking and bioavailability. The emergence of inter-organellar membrane contact sites, as well as intracellular vesicles derived from various organelles, have raised the prospect that heme can be trafficked through hydrophobic channels. In this review, we aim to focus on heme delivery without deliverers - an alternate paradigm for the regulation of heme homeostasis through chaperone-less pathways for heme trafficking.


Assuntos
Heme/metabolismo , Homeostase/efeitos dos fármacos , Lipídeos/química , Transporte Proteico/genética , Citotoxinas/farmacologia , Heme/química , Homeostase/genética , Ferro/química , Ferro/metabolismo , Metais/química , Chaperonas Moleculares/química , Solubilidade/efeitos dos fármacos
11.
Nat Commun ; 11(1): 5736, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-33184256

RESUMO

Highly charged intrinsically disordered proteins can form complexes with very high affinity in which both binding partners fully retain their disorder and dynamics, exemplified by the positively charged linker histone H1.0 and its chaperone, the negatively charged prothymosin α. Their interaction exhibits another surprising feature: The association/dissociation kinetics switch from slow two-state-like exchange at low protein concentrations to fast exchange at higher, physiologically relevant concentrations. Here we show that this change in mechanism can be explained by the formation of transient ternary complexes favored at high protein concentrations that accelerate the exchange between bound and unbound populations by orders of magnitude. Molecular simulations show how the extreme disorder in such polyelectrolyte complexes facilitates (i) diffusion-limited binding, (ii) transient ternary complex formation, and (iii) fast exchange of monomers by competitive substitution, which together enable rapid kinetics. Biological polyelectrolytes thus have the potential to keep regulatory networks highly responsive even for interactions with extremely high affinities.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Polieletrólitos/química , Cinética , Espectroscopia de Ressonância Magnética , Chaperonas Moleculares/química , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Precursores de Proteínas/química , Coloração e Rotulagem , Timosina/análogos & derivados
12.
J Comput Aided Mol Des ; 34(12): 1237-1259, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33034007

RESUMO

Computational protein-ligand docking is well-known to be prone to inaccuracies in input receptor structures, and it is challenging to obtain good docking results with computationally predicted receptor structures (e.g. through homology modeling). Here we introduce a fragment-based docking method and test if it reduces requirements on the accuracy of an input receptor structures relative to non-fragment docking approaches. In this method, small rigid fragments are docked first using AutoDock Vina to generate a large number of favorably docked poses spanning the receptor binding pocket. Then a graph theory maximum clique algorithm is applied to find combined sets of docked poses of different fragment types onto which the complete ligand can be properly aligned. On the basis of these alignments, possible binding poses of complete ligand are determined. This docking method is first tested for bound docking on a series of Cytochrome P450 (CYP450) enzyme-substrate complexes, in which experimentally determined receptor structures are used. For all complexes tested, ligand poses of less than 1 Å root mean square deviations (RMSD) from the actual binding positions can be recovered. Then the method is tested for unbound docking with modeled receptor structures for a number of protein-ligand complexes from different families including the very recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protease. For all complexes, poses with RMSD less than 3 Å from actual binding positions can be recovered. Our results suggest that for docking with approximately modeled receptor structures, fragment-based methods can be more effective than common complete ligand docking approaches.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/efeitos dos fármacos , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Proteínas não Estruturais Virais/efeitos dos fármacos , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Ligantes , Modelos Químicos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
13.
Nucleic Acids Res ; 48(16): 9273-9284, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32761152

RESUMO

Nucleic acid-binding proteins of the Sac10b family, also known as Alba, are widely distributed in Archaea. However, the physiological roles of these proteins have yet to be clarified. Here, we show that Sis10b, a member of the Sac10b family from the hyperthermophilic archaeon Sulfolobus islandicus, was active in RNA strand exchange, duplex RNA unwinding in vitro and RNA unfolding in a heterologous host cell. This protein exhibited temperature-dependent binding preference for ssRNA over dsRNA and was more efficient in RNA unwinding and RNA unfolding at elevated temperatures. Notably, alanine substitution of a highly conserved basic residue (K) at position 17 in Sis10b drastically reduced the ability of this protein to catalyse RNA strand exchange and RNA unwinding. Additionally, the preferential binding of Sis10b to ssRNA also depended on the presence of K17 or R17. Furthermore, normal growth was restored to a slow-growing Sis10b knockdown mutant by overproducing wild-type Sis10b but not by overproducing K17A in this mutant strain. Our results indicate that Sis10b is an RNA chaperone that likely functions most efficiently at temperatures optimal for the growth of S. islandicus, and K17 is essential for the chaperone activity of the protein.


Assuntos
Proteínas Arqueais/genética , Proteínas de Ligação a DNA/genética , Chaperonas Moleculares/genética , RNA/genética , Archaea/genética , Proteínas Arqueais/química , Proteínas de Ligação a DNA/química , Chaperonas Moleculares/química , Ligação Proteica/genética , Dobramento de RNA/genética , RNA de Cadeia Dupla/genética , Sulfolobus/química , Sulfolobus/genética
14.
Nature ; 584(7822): 630-634, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32814900

RESUMO

Integral membrane proteins are encoded by approximately 25% of all protein-coding genes1. In eukaryotes, the majority of membrane proteins are inserted, modified and folded at the endoplasmic reticulum (ER)2. Research over the past several decades has determined how membrane proteins are targeted to the ER and how individual transmembrane domains (TMDs) are inserted into the lipid bilayer3. By contrast, very little is known about how multi-spanning membrane proteins with several TMDs are assembled within the membrane. During the assembly of TMDs, interactions between polar or charged amino acids typically stabilize the final folded configuration4-8. TMDs with hydrophilic amino acids are likely to be chaperoned during the co-translational biogenesis of membrane proteins; however, ER-resident intramembrane chaperones are poorly defined. Here we identify the PAT complex, an abundant obligate heterodimer of the widely conserved ER-resident membrane proteins CCDC47 and Asterix. The PAT complex engages nascent TMDs that contain unshielded hydrophilic side chains within the lipid bilayer, and it disengages concomitant with substrate folding. Cells that lack either subunit of the PAT complex show reduced biogenesis of numerous multi-spanning membrane proteins. Thus, the PAT complex is an intramembrane chaperone that protects TMDs during assembly to minimize misfolding of multi-spanning membrane proteins and maintain cellular protein homeostasis.


Assuntos
Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/metabolismo , Biossíntese de Proteínas , Sequência de Aminoácidos , Asparagina/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Chaperonas Moleculares/química , Complexos Multiproteicos/química , Mutação , Proteínas Nucleares/metabolismo , Ligação Proteica , Dobramento de Proteína , Subunidades Proteicas/metabolismo , Especificidade por Substrato
15.
Nucleic Acids Res ; 48(15): e90, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32609809

RESUMO

Specific genomic functions are dictated by macromolecular complexes (MCs) containing multiple proteins. Affinity purification of these complexes, often using antibodies, followed by mass spectrometry (MS) has revolutionized our ability to identify the composition of MCs. However, conventional immunoprecipitations suffer from contaminating antibody/serum-derived peptides that limit the sensitivity of detection for low-abundant interacting partners using MS. Here, we present AptA-MS (aptamer affinity-mass spectrometry), a robust strategy primarily using a specific, high-affinity RNA aptamer against Green Fluorescent Protein (GFP) to identify interactors of a GFP-tagged protein of interest by high-resolution MS. Utilizing this approach, we have identified the known molecular chaperones that interact with human Heat Shock Factor 1 (HSF1), and observed an increased association with several proteins upon heat shock, including translation elongation factors and histones. HSF1 is known to be regulated by multiple post-translational modifications (PTMs), and we observe both known and new sites of modifications on HSF1. We show that AptA-MS provides a dramatic target enrichment and detection sensitivity in evolutionarily diverse organisms and allows identification of PTMs without the need for modification-specific enrichments. In combination with the expanding libraries of GFP-tagged cell lines, this strategy offers a general, inexpensive, and high-resolution alternative to conventional approaches for studying MCs.


Assuntos
Aptâmeros de Nucleotídeos/química , Fatores de Transcrição de Choque Térmico/química , Substâncias Macromoleculares/isolamento & purificação , Espectrometria de Massas , Aptâmeros de Nucleotídeos/genética , Proteínas de Fluorescência Verde/genética , Fatores de Transcrição de Choque Térmico/genética , Histonas/química , Humanos , Imunoprecipitação , Substâncias Macromoleculares/química , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Peptídeos/química , Ligação Proteica , Processamento de Proteína Pós-Traducional
16.
Mol Biol (Mosk) ; 54(2): 300-307, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32392200

RESUMO

The thermal stability of protein enzymes is determined in vitro by measuring the enzymatic activity during incubation at constant temperature. Refolding of thermal inactivated enzymes is carried out both in vitro and in vivo, in the presence of chaperones, usually at temperature optimal for the particular enzyme for the manifestation of enzymatic activity. In the present work thermal stability of enzymes in vitro (using purified preparations) and in vivo (directly in the bacterial cell) has been determined. Bacterial luciferases of Aliivibrio fischeri, Photobacterium leiognathi and Photorhabdus luminescens as protein substrates have been used. It is shown that the thermal stability of the P. luminescens and P. leiognathi luciferases in vivo in the Escherichia coli MG1655 dnaK^(+) and PK202 ΔdnaKJ14 strains is considerable higher than the thermal stability of "cell-free extract" luciferases. When an uncoupler of oxidative phosphorylation the carbonyl-cyanide-3-chlorophenylhydrazone (CCCP) that reduce the intracellular concentration of ATP to a minimum level, and the volatile hydrophobic substance (-)-Limonene (C10H16) as an inhibitor of chaperone-dependent refolding are added to the medium, the thermal stability of luciferases reduces almost to the level which is characteristic for the purified protein preparation. It is shown that the ATP-dependent chaperones ClpA and ClpB are essential for the increase of thermostability of luciferases in bacterial cells. Also, it is shown that the DnaKJE-dependent refolding of thermoinactivated luciferases is practically absent if the protonophore СССР or the hydrophobic substance (-)-Limonene was added to the bacterial suspension. Taking the data presented in this paper into account, it is necessary to consider the presence in bacterial cells of two different groups of ATP-dependent chaperones: 1st group (DnaKJE, GroEL/ES) is able to conduct the refolding both at low temperature after protein thermal inactivation and at high temperature at which protein thermal inactivation occurs; 2nd group (ClpA,ClpB, and possibly still unknown chaperones) is unable to conduct the standard refolding (i.e. at low temperature), but capable due to the hydrolysis energy of ATP of maintaining nonequilibrium stabilization of protein native forms at high temperature.


Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Chaperonas Moleculares/química , Dobramento de Proteína , Endopeptidase Clp , Estabilidade Proteica , Temperatura
17.
Adv Exp Med Biol ; 1194: 351-358, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32468551

RESUMO

Protein homeostasis is a dynamic network that plays a pivotal role in systems' maintenance within a cell. This quality control system involves a number of mechanisms regarding the process of protein folding. Chaperones play a critical role in the folding, refolding, and unfolding of proteins. Aggregation of misfolded proteins is a common characteristic of neurodegenerative diseases. Chaperones act in a variety of pathways in this critical interplay between protein homeostasis network and misfolded protein's load. Moreover, ER stress-induced cell death mechanisms (such as the unfolded protein response) are activated as a response. Therefore, there is a critical balance in the accumulation of misfolded proteins and ER stress response mechanisms which can lead to cell death. Our focus is in understanding the different mechanisms that govern ER stress signaling in health and disease in order to represent the regulation of protein homeostasis and balance of protein synthesis and degradation in the ER. Our proposed model describes, using hybrid modeling, the function of chaperones' machinery for protein folding.


Assuntos
Modelos Biológicos , Chaperonas Moleculares , Dobramento de Proteína , Humanos , Chaperonas Moleculares/química , Doenças Neurodegenerativas/fisiopatologia , Biossíntese de Proteínas , Proteínas/metabolismo , Deficiências na Proteostase , Transdução de Sinais , Resposta a Proteínas não Dobradas
18.
Nat Commun ; 11(1): 1909, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32312993

RESUMO

Peptide exchange technologies are essential for the generation of pMHC-multimer libraries used to probe diverse, polyclonal TCR repertoires in various settings. Here, using the molecular chaperone TAPBPR, we develop a robust method for the capture of stable, empty MHC-I molecules comprising murine H2 and human HLA alleles, which can be readily tetramerized and loaded with peptides of choice in a high-throughput manner. Alternatively, catalytic amounts of TAPBPR can be used to exchange placeholder peptides with high affinity peptides of interest. Using the same system, we describe high throughput assays to validate binding of multiple candidate peptides on empty MHC-I/TAPBPR complexes. Combined with tetramer-barcoding via a multi-modal cellular indexing technology, ECCITE-seq, our approach allows a combined analysis of TCR repertoires and other T cell transcription profiles together with their cognate antigen specificities in a single experiment. The new approach allows TCR/pMHC interactions to be interrogated easily at large scale.


Assuntos
Proteínas de Transporte/química , Antígenos de Histocompatibilidade Classe I/química , Proteínas de Membrana Transportadoras/química , Chaperonas Moleculares/química , Peptídeos/química , Domínios e Motivos de Interação entre Proteínas , Alelos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Biblioteca Gênica , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunidade Celular , Imunoquímica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Linfócitos T
19.
Nat Struct Mol Biol ; 27(4): 363-372, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231288

RESUMO

Protein phase separation drives the assembly of membraneless organelles, but little is known about how these membraneless organelles are maintained in a metastable liquid- or gel-like phase rather than proceeding to solid aggregation. Here, we find that human small heat-shock protein 27 (Hsp27), a canonical chaperone that localizes to stress granules (SGs), prevents FUS from undergoing liquid-liquid phase separation (LLPS) via weak interactions with the FUS low complexity (LC) domain. Remarkably, stress-induced phosphorylation of Hsp27 alters its activity, leading Hsp27 to partition with FUS LC to preserve the liquid phase against amyloid fibril formation. NMR spectroscopy demonstrates that Hsp27 uses distinct structural mechanisms for both functions. Our work reveals a fine-tuned regulation of Hsp27 for chaperoning FUS into either a polydispersed state or a LLPS state and suggests an essential role for Hsp27 in stabilizing the dynamic phase of stress granules.


Assuntos
Proteínas de Choque Térmico HSP27/química , Chaperonas Moleculares/química , Proteína FUS de Ligação a RNA/química , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/isolamento & purificação , Humanos , Extração Líquido-Líquido , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Fosforilação , Ligação Proteica/genética , Domínios Proteicos/genética , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Estresse Fisiológico/genética
20.
Prog Nucl Magn Reson Spectrosc ; 116: 56-84, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32130959

RESUMO

A major goal in structural biology is to unravel how molecular machines function in detail. To that end, solution-state NMR spectroscopy is ideally suited as it is able to study biological assemblies in a near natural environment. Based on methyl TROSY methods, it is now possible to record high-quality data on complexes that are far over 100 kDa in molecular weight. In this review, we discuss the theoretical background of methyl TROSY spectroscopy, the information that can be extracted from methyl TROSY spectra and approaches that can be used to assign methyl resonances in large complexes. In addition, we touch upon insights that have been obtained for a number of challenging biological systems, including the 20S proteasome, the RNA exosome, molecular chaperones and G-protein-coupled receptors. We anticipate that methyl TROSY methods will be increasingly important in modern structural biology approaches, where information regarding static structures is complemented with insights into conformational changes and dynamic intermolecular interactions.


Assuntos
Espectroscopia de Ressonância Magnética , Animais , Humanos , Marcação por Isótopo , Proteínas de Membrana/química , Metilação , Modelos Moleculares , Chaperonas Moleculares/química
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